Investigations on hepatitis B virus (HBV) and related animal viruses, particularly duck hepatitis B virus (DHBV), have provided a detailed model of the hepadnavirus replication cycle. However, the mechanism responsible for the conversion of the relaxed circular (RC) viral DNA genome into covalently closed circular (CCC) DNA, the template for viral RNA transcription, is not yet known. Likewise, the mechanism regulating amplification of CCC DNA in mammalian hepadnaviruses is still elusive. Research from our laboratory showed that the conversion of DHBV RC into CCC DNA occurs independently of viral enzymatic functions, and requires instead cellular DNA repair enzymes that are resistant to aphidicolin and dideoxynucleotides. Consistent with these results, we have obtained compelling evidence that the Y DNA polymerase kappa, central to nucleotide excision repair (NER), is required for DHBV CCC DNA synthesis. We propose a novel hypothesis stipulating that HBV, and DHBV, use the cellular nucleotide excision repair machinery for the conversion of RC DNA into CCC DNA. The hypothesis will be investigated through the following specific aims: Aim 1. Identification of cellular factors required for the conversion of HBV and DHBV RC into CCC DNA. The purpose of this aim is to determine if polymerase is also required for HBV CCC DNA synthesis and to determine if the endonucleases and DNA ligases involved in NER play a role in the conversion of DHBV, as well as HBV RC to CCC DNA. Aim 2. Identification of intermediates of minus and plus strand DNA. We propose experiments to identify DNA intermediates predicted to accumulate in cells lacking pol and investigate whether the repair of minus and plus strand DNA occurs by the same or different mechanisms. This proposal will fill a major gap in our understanding of hepadnavirus biology, the mechanism of CCC DNA synthesis, and reveal a completely novel strategy used by an animal virus to replicate DNA. The information gained from our research into the mechanism of CCC DNA synthesis could be exploited for the identification of novel cellular genes that might provide targets for future antiviral therapies, required for the cure of over 400 million HBV infected people. Moreover, the aims of the application represent an exploratory effort that will set the stage for future investigations into he mechanism used by HBV to recruit the NER system and on the mechanism controlling the observed species specificity of HBV CCC DNA synthesis. PUBLIC HEALTH RELEVANCE: Over 400 million people are currently infected with hepatitis B virus and are at significantly elevated risk for the development of liver cancer. Our research into the mechanism of CCC DNA synthesis is directed at identification of cellular genes that represent novel targets for future antiviral therapies that, in combination with existing drugs, might cure HBV infections.